The Gram-negative bacterium Francisella tularensis subsp. tularensis, the causative agent of tularemia, is considered a potentially dangerous biological weapon. This is due to the organism's extreme infectivity via the aerosol route and the severity of tularemia. The fastidious nature of the bacterium, the requirement that it be handled using biosafety level three (BSL3) containment, and the paucity of genetic tools have hindered research on this organism. Thus, compared to many other bacterial pathogens, little is known about F. tularensis subsp. tularensis physiology, genetics, and pathogenesis. Previous studies have used the attenuated live vaccine strain (LVS) derived from F. tularensis subsp. holarctica, or the rarely pathogenic F. tularensis subsp. novicida. The goal of this proposal is to identify secreted proteins that are important in the pathogenesis of the virulent F. tularensis subsp. tularensis. We will use recently developed genetic tools to identify and disrupt genes encoding secreted proteins with the immediate goal of identifying proteins required for intracellular survival and replication in mouse macrophages. We anticipate that this work will identify several proteins of interest and will allow us to formulate hypotheses for the basis of further work. The work proposed in this application would contribute to the research goals of the NIAID biodefense research agenda for CDC category A agents such as Francisella tularensis. These immediate goals include; 1) the development of genetic systems to correlate differences in pathogenesis and virulence and 2) increased diversity of expertise in the study of tularemia. [unreadable] The research in this proposal will provide additional genetic methods for the study of F. tularensis biology and identify factors important for the pathogenesis of tularemia disease. This work may also contribute to better diagnostics, vaccine and drug development. Because of F. tularensis is a select agent and could be used a biological weapon, this research could potentially have a significant impact on public health in the future. [unreadable] [unreadable] [unreadable]